Human T-cell leukemia virus type 1 (HTLV-1) is linked to the genesis of adult T-cell leukemia (ATL), an aggressive CD4+CD25+ malignancy. Tax is an oncoprotein encoded in the pX region of the HTLV-1 genome that regulates viral and cellular gene expression and promotes cell transformation, in part, by constitutive activation of the NF-?B transcription factor. Tax requires NF-?B for the immortalization of primary T cells and HTLV-1 transformed cells are dependent on NF-?B for their survival, thereby providing a strong rationale for studying the mechanisms used by Tax to activate NF-?B. Tax activates the I?B kinase (IKK) and NF-?B persistently by interacting with the IKK regulatory subunit NEMO, however the underlying mechanisms remain poorly understood. Our collective studies reinforce the notion that Tax hijacks the host ubiquitin machinery to activate NF-?B. Tax is polyubiquitinated via Lys63 (K63)-linked polyubiquitin chains and requires the E2 ubiquitin conjugating enzyme Ubc13 for Tax ubiquitination, NEMO binding and NF-?B activation. In our preliminary studies we have conducted ubiquitin proteomics screening which has identified the anti-apoptotic protein Mcl-1 as a downstream target of Tax and the E3 ligase TRAF6. Tax-induced K63-linked polyubiquitination of Mcl-1 endowed Mcl-1 with enhanced stability and prevented its degradation by the chemotherapy drug etoposide. Using a yeast two-hybrid screen we have also identified the E3/E4 enzyme UBE4B as a novel Tax interacting protein that may regulate Tax K63-linked polyubiquitination and NF-?B activation. We have also demonstrated that Tax has usurped the adaptor function of TAX1BP1, a scaffold molecule for the NF-?B inhibitor and ubiquitin-editing enzyme A20, to promote NF-?B signaling. In this competitive renewal, we will continue to establish the mechanisms by which Tax hijacks ubiquitin machinery components to promote persistent NF-?B activation and cell survival. The specific aims of the proposal are to: (1) determine the mechanisms of Mcl-1 stabilization by Tax; (2) determine the role of the host ubiquitin machinery in Tax-induced NF-?B activation and (3) determine the role of TAX1BP1 in Tax activation of NF-?B and leukemogenesis. These studies will elucidate how a retroviral oncoprotein hijacks host ubiquitination machinery to inhibit cell death and promote cell transformation.